The objective of the proposed research is to understand, in chemical terms, the biological activities of three erythrocyte hemeproteins- cytochrome b5, green cyanide-binding hemeprotein, and hemeprotein 559, - and an erythrocyte pink copper protein. Homogeneous proteins from human and bovine erythrocytes will be subjected to chemical, physical, immunological, and catalytic studies. The primary structure, physical properties, and catalytic properties of erythrocyte cytochrome b5 will be determined and compared to liver microsomal cytochrome b5. The role of cytochrome b5 in the reduction of methemoglobin will be studied kinetically using reconstituted systems and crude hemolysates of red cells from normal donors and patients with congential methemoglobinemia. We will attempt to establish the structure of the unique prosthetic cyanide-binding hemeprotein. The reaction of this hemeprotein with oxygen and hydrogen peroxide will be studied and a search will be made for its reducing system. Such studies will hopefully allow us to assign a biological activity to the protein and to detect the protein in other tissues. The chemical, physical, and immunological properties of hemeprotein 559 will be studied in order to establish whether this protein is a degraded form of cytochrome P-450. The pink copper protein of erythrocytes will be studied to determine the nature of its chromophore and to characterize and understand the significance of its oxidase activity. The results of these studies are important in terms of understanding the structure of the unique prosthetic group of the green hemeprotein, the mechanism of methemoglobin reduction, and the metabolism of erythrocytes in general. Furthermore, these studies may provide a better understanding of red blood cell maturation and aging, the defects of erythroleukemia, methemoglobinemia, and multiple myeloma, and the problems of blood storage, copper deficiency, and copper toxicity. BIBLIOGRAPHIC REFERENCES: Hultquist, D.E., Dean, R.T. and Reed, D.W. (1976) Isolation and Characterization of the Unique Prosthetic Group of a Green Hemeprotein from Human Erythrocytes, J. Biol. Chem. 251, 3927-3932. Slaughter, S.R., Hultquist, D.E. (1977) Demonstration of Microsomal Cytochrome b5, Cytochrome P-450 in Friend Erythroleukemia (FL) Cells, Fed. Proc. 36.